Plant crops are a desirable host for the production of a range of metabolic products including modified vegetable oils, polyhydroxyalkanoates, amino acids, modified lignins, modified starches and nutraceutical products. Very often production of these new products requires the expression of two or more transgenes encoding two or more polypeptides having enzyme activities. It is desirable to be able to express two or more transgenes at a point in the developmental cycle of the plant to maximize the formation of a desired product. In other cases it is desirable to be able to switch on the expression of a metabolic pathway to mitigate negative impacts on plant growth or yield caused by the polypeptides or products of the metabolic pathways.
The development of agricultural systems, in which bioplastics can be produced economically and sustainably in green plants, has the potential to not only dramatically lower the cost of bioplastics, but to sequester CO2.
Polyhydroxyalkanoates (PHAs) are a family of biodegradable biopolymers that can be produced in plants. The desired commercial target of PHA production in plants is 7.5% to 15% dry weight (dwt) (Y. Poirier and K. J. Gruys, in Biopolyesters, Y. Doi, A. Steinbuchel Eds. (Wiley-VCH, Weinheim; 2002), pp. 401-435). To date, PHB has been successfully produced in the following plant species: Arabidopsis thaliana, Brassica napus and Zea mays (C. Nawrath et al. Proc. Natl. Acad. Sci. USA 91, 12760 (1994); K. Bohmert et al., Planta 211, 841 (2000); K. L. Houmiel, et al., Planta 209, 547 (1999); Y. Poirier and K. J. Gruys, in Biopolyesters, Y. Doi, A. Steinbuchel Eds. (Wiley-VCH, Weinheim; 2002), pp. 401-435). However, plants producing in excess of 3% dwt PHB often develop a chlorotic phenotype and/or do not achieve full size (Bohmert, K. et al., in Molecular Biology and Biotechnology of Plant Organelles. H. Daniell, C. D. Chase Eds. (Kluwer Academic Publishers, Netherlands; 2004, pp. 559-585). These factors resulted in low total polymer yields and represent a major obstacle to the plant-based production of PHA. Attempts to overcome the problem of low total yields using an inducible promoter to control the expression of a single gene in the PHB pathway have yielded high levels of leaky polymer production in un-induced plants such that plants were still stunted (Bohmert et. al. Plant Physiol. 128(4): 1282-90. (2002)).
It is therefore an object of the invention to provide vectors or constructs for the inducible expression of two or more genes encoding two or more enzyme activities in a metabolic pathway in plant crops.
It is a further object of the invention to transform plants with these vectors or constructs and induce the coordinated expression of two or more transgenes encoding two or more enzyme activities required for efficient formation of the desired product in the host plant, such as a biopolymer, a novel oil, a modified lignin, a modified starch or a nutraceutical while limiting detrimental effects that can be associated with constitutive expression of the transgene encoded enzymes in pathways, such as those leading to enhanced formation of the desired product.
It is a still further object of this invention to induce these genes by the foliar or root application of a chemical inducing agent such that the genes are expressed, and the flow of metabolic intermediates is channeled through the appropriate metabolic pathway to enhance the production of a product of that pathway.
It is a further object of this invention to provides methods of application of the chemical inducing agent by foliar or root application at the optimum time during the plant growth cycle to enhance the production of the desired product and to harvest the plant material and recover the product of interest.